Background: Patients with locally advanced esophageal cancer who are treated with trimodality therapy have a high recurrence rate. Preclinical evidence suggests that inhibition of cyclooxygenase 2 (COX2) increases the effectiveness of chemoradiation, and observational studies in humans suggest that COX-2 inhibition may reduce esophageal cancer risk. This trial tested the safety and efficacy of combining a COX2 inhibitor, celecoxib, with neoadjuvant irinotecan/cisplatin chemoradiation. Methods: This single arm phase 2 trial combined irinotecan, cisplatin, and celecoxib with concurrent radiation therapy. Patients with stage IIA-IVA esophageal cancer received weekly cisplatin 30 mg/m(2) plus irinotecan 65 mg/m(2) on weeks 1, 2, 4, and 5 concurrently with 5040 cGy of radiation therapy. Celecoxib 400 mg was taken orally twice daily during chemoradiation, up to 1 week before surgery, and for 6 months following surgery. Results: Forty patients were enrolled with stage IIa (30 %), stage IIb (20 %), stage III (22.5 %), and stage IVA (27.5 %) esophageal or gastroesophageal junction cancer (AJCC, 5th Edition). During chemoradiation, grade 3-4 treatment-related toxicity included dysphagia (20 %), anorexia (17.5 %), dehydration (17.5 %), nausea (15 %), neutropenia (12.5 %), diarrhea (10 %), fatigue (7.5 %), and febrile neutropenia (7.5 %). The pathological complete response rate was 32.5 %. The median progression free survival was 15.7 months and the median overall survival was 34.7 months. 15 % (n = 6) of patients treated on this study developed brain metastases. Conclusions: The addition of celecoxib to neoadjuvant cisplatin-irinotecan chemoradiation was tolerable; however, overall survival appeared comparable to prior studies using neoadjuvant cisplatin-irinotecan chemoradiation alone. Further studies adding celecoxib to neoadjuvant chemoradiation in esophageal cancer are not warranted.

Cancer is a stem cell disease and populations of cancer stem cells (CSCs) are evident in many cancer types. CSCs exhibit similarities to normal embryonic and adult stem cells: they are able to self-renew and have the potential to give rise to a diverse array of differentiated progeny. CSCs are responsible for driving tumourigenesis and metastasis, and are inherently resistant to chemotherapy and radiotherapy. This cell population is enriched after treatment and, as a result of their tumourigenic capability, can re-populate tumour growth resulting in patient relapse, often with increased chemotherapeutic resistance. Increasing evidence supports that only by targeting this population of cells will a cure for cancer be possible. Hence, it is essential to identify pathways within CSC populations that can be targeted therapeutically. Cyclooxygenase-2 (COX-2) is an enzyme associated with inflammation and disease, and is upregulated in many cancers types. The COX-2 / prostaglandin E2 (PGE2) signalling pathway is associated with increased tumour growth, metastasis, immune evasion and overall worse patient prognosis. Recent evidence has identified that COX-2 is further upregulated in CSC populations isolated from cancer cell lines. Previously, we have shown that inhibition of COX-2 reduces CSC sphere-forming ability, a characteristic of stem cell self-renewal, suggesting a role for COX-2 in maintaining CSC populations. This work was carried out in both human and canine osteosarcoma cell lines with similar results. Cancer in dogs is a major health concern among an aging pet population. Many cancer types exhibit similarities between these species, suggesting that naturally occurring canine cancer may be a potential model for the human disease. The aim of this PhD project was to investigate the role of COX-2 in CSCs in a comparative cancer study. CSCs that express stem cell markers have been isolated from a panel of canine and human cancer cell lines including, mammary carcinoma and transitional cell carcinoma of the urinary bladder. CSCs over-express COX-2 compared to non-CSCs, therefore to determine the role of COX-2 in CSC biology the selective COX-2 inhibitor mavacoxib, a non-steroidal anti-inflammatory drug currently licenced for treating osteoarthritis in dogs, was utilised. Our results demonstrate that inhibiting COX-2 has a multifaceted impact on CSC biology, including reducing self-renewal capacity, clonogenicity, proliferation, migration, invasion and in vivo tumourigenicity. To confirm that mavacoxib is mediating these CSC-specific effects via inhibition of COX-2 rather than through unknown off-target effects, we generated canine specific-small interfering RNA to specifically reduce gene expression of COX-2. Our results confirm that mavacoxib exerts its anti-tumour effects via inhibition of COX-2. This project has highlighted a plethora of CSC-specific COX-2 effects, and to gain further insight we compared the global gene expression profiles of CSCs compared to non- CSCs isolated from a canine bladder carcinoma cell line. This data revealed that both mavacoxib and COX-2 specific siRNA target similar pathways within the two cell populations, confirming that mavacoxib exerts its effects in a COX-2 dependent manner. Interestingly, mavacoxib reduced the expression of a number of stemness related genes in the CSC population, including NOTCH and Wnt, suggesting that mavacoxib can inhibit CSC related pathways. Our overall results are comparable between canine and human cancer cell lines supporting the concept of naturally occurring tumours in dogs as models for the human disease. In conclusion, COX-2 plays an important role not only in maintaining CSC populations but also in their function, and targeting COX-2 in CSCs may provide therapeutic benefit.

Advances in next-generation sequencing and ribosomal profiling methods highlight that the proteome is likely orders of magnitude larger than previously thought. This expansion potentially occurs through translational recoding, a process that results in the expression of multiple variations of a protein from a single messenger RNA. Our laboratory demonstrated that cyclooxygenase-3/1b (COX-3/1b), a frameshifted, intron-1-retaining, alternative splice variant from the COX-1 gene, is multiply recoded, which results in the translation of at least seven different COX-3 proteins. Two of the recoded COX-3 proteins that we identified are active prostaglandin synthases and are inhibited by non-steroidal anti-inflammatory drugs (NSAIDs). Here we show that the other non-prostaglandin-generating recoded COX-3 proteins perform new roles in innate immunity, a process in which COX are known to generally function. Our analyses determined that these recoded COX-3 proteins bind at or near the amino-terminal region of ATG9a, a critical regulator of both canonical (i.e. digestive autophagy associated with mTORc inhibition and nutrient deprivation) and non-canonical (i.e. xenophagy involved in the innate immune response to invading organisms) autophagy. We further show that this process requires mTORc signaling activity, which opposes the digestive pathway. As a final confirmation of the biological relevance of these recoded COX-3 proteins and their central role in xenophagy, we demonstrate that expression of these COX-3 proteins in an encephalomyocarditis virus infection model system differentially affects infectious virion production. These COX-3 proteins also associate with recoded cytosolic nucleobindin around large, innate immune-related, large LC3-II positive structures (LLPSs). Through mutagenizing catalytic residues of recoded COX-3 proteins and drug assays, we determine LLPS formation is dependent on oxylipin generation.

Abstract not available

Developmental exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes severe defects in the cardiovascular system. However, the effects of acute AhR agonist exposure on the adult fish cardiovascular system as well as the genes mediating developmental AhR-induced deformities remain unclear. In this thesis, two studies were carried out to address these issues. Before experiments could begin, methods for quantitative real-time reverse transcriptase polymerase chain reaction (rtrt-PCR) as well as larval exposure and rearing were developed, validated, and optimized.<p> Following method development, a series of experiments was performed on adult zebrafish (<i>Danio rerio</i>) to assess how expression of cytochrome P450 (CYP) and cyclooxygenase (COX) enzyme mRNA in hepatic and vascular tissues is altered after intraperitoneal injection of AhR agonists benzo(a)pyrene (BaP) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone and in combination with the purported AhR antagonists resveratrol (Res) or alpha-naphthoflavone (ANF). Both TCDD and BaP induced similar patterns of gene expression in arteries, although with different efficacies, and had slightly different effects in hepatic tissues. Resveratrol was generally without effect in all treatment groups and tissues with the exception of reducing TCDD-induced CYP1C2 in vascular tissues. In contrast, ANF antagonized TCDD- and BaP-induced changes, as well as reduced baseline gene expression in liver. However, in arteries, ANF alone acted as an agonist to increase expression of several of the genes investigated.<p> The second series of experiments involved zebrafish eggs aqueously exposed to BaP or TCDD alone and in combination with Res or ANF. Whole larvae CYP and COX isoform mRNA expression was quantified at 5 and 10 days post-fertilization (dpf), then correlated with developmental phenotype. Both TCDD and BaP caused concentration-dependent AhR-associated deformities with a significant increase in mortalities by 10 dpf and increased CYP1A mRNA expression, while TCDD alone decreased CYP1C2 expression. BaP/ANF co-exposure exhibited the highest rate of deformities and mortalities at both 5 and 10 dpf, caused marked alterations in cardiac and vascular morphology at 10 dpf, and increased CYP1A expression. Furthermore, ANF exhibited additive agonistic effects on gene expression with both BaP and TCDD. Correlation analyses revealed that gene expression at 5 dpf, but not 10 dpf, was strongly linked to abnormal cardiac and vascular phenotypes at 10 dpf with several genes related to cardiac development and one primary gene linked to vascular development.

Developmental exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes severe defects in the cardiovascular system. However, the effects of acute AhR agonist exposure on the adult fish cardiovascular system as well as the genes mediating developmental AhR-induced deformities remain unclear. In this thesis, two studies were carried out to address these issues. Before experiments could begin, methods for quantitative real-time reverse transcriptase polymerase chain reaction (rtrt-PCR) as well as larval exposure and rearing were developed, validated, and optimized.<p> Following method development, a series of experiments was performed on adult zebrafish (<i>Danio rerio</i>) to assess how expression of cytochrome P450 (CYP) and cyclooxygenase (COX) enzyme mRNA in hepatic and vascular tissues is altered after intraperitoneal injection of AhR agonists benzo(a)pyrene (BaP) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alone and in combination with the purported AhR antagonists resveratrol (Res) or alpha-naphthoflavone (ANF). Both TCDD and BaP induced similar patterns of gene expression in arteries, although with different efficacies, and had slightly different effects in hepatic tissues. Resveratrol was generally without effect in all treatment groups and tissues with the exception of reducing TCDD-induced CYP1C2 in vascular tissues. In contrast, ANF antagonized TCDD- and BaP-induced changes, as well as reduced baseline gene expression in liver. However, in arteries, ANF alone acted as an agonist to increase expression of several of the genes investigated.<p> The second series of experiments involved zebrafish eggs aqueously exposed to BaP or TCDD alone and in combination with Res or ANF. Whole larvae CYP and COX isoform mRNA expression was quantified at 5 and 10 days post-fertilization (dpf), then correlated with developmental phenotype. Both TCDD and BaP caused concentration-dependent AhR-associated deformities with a significant increase in mortalities by 10 dpf and increased CYP1A mRNA expression, while TCDD alone decreased CYP1C2 expression. BaP/ANF co-exposure exhibited the highest rate of deformities and mortalities at both 5 and 10 dpf, caused marked alterations in cardiac and vascular morphology at 10 dpf, and increased CYP1A expression. Furthermore, ANF exhibited additive agonistic effects on gene expression with both BaP and TCDD. Correlation analyses revealed that gene expression at 5 dpf, but not 10 dpf, was strongly linked to abnormal cardiac and vascular phenotypes at 10 dpf with several genes related to cardiac development and one primary gene linked to vascular development.

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The purpose of this study was to assess the prognostic significance and clinical correlations of cyclooxgenase-2 expression (COX) in a cohort of patients treated with radiation (RT) for post-mastectomy chest wall relapse (PMCWR). Between 1975 and 1999, 113 patients were treated for isolated PMCWR. All patients were treated with biopsy and/or excision of the CWR followed by RT. Median follow-up was 10 years. All clinical data including demographics, pathology, staging, receptor status, HER-2/neu status, and adjuvant therapy were entered into a computerized database. Paraffin-embedded CWR specimens were retrieved from 42 patients, of which 38 were evaluated, created into a tissue microarray, stained by immunohistochemical methods for COX, and graded 0-3+. A score of 2-3+ was considered positive. Overall survival from original diagnosis for the entire cohort was 44% at 10 years. Survival rate after chest wall recurrence was 28% at 10 years. The distant metastasis-free survival rate after CWR was 40% at 10 years. Local-regional control of disease was achieved in 79% at 10 years after CWR. COX was considered positive in 13 of 38 cases. COX was inversely correlated with ER (p= .045) and PR (p = .028), and positively correlated with HER-2/neu (p =.003). COX was also associated with a shorter time to PMCWR. The distant metastasis-free rate for COX negative patients was 70% at 10 years, compared with 31% at 10 years for COX-2 positive patients (p = 0.029). COX positive had a poorer local-regional progression-free rate of 19% at 10 years, compared with 81% at 10 years for COX negative (p = 0.003). Outcome following RT for PMCWR is relatively poor. Positive COX correlated with other markers of poor outcome including a shorter time to local relapse, negative ER/PR and positive Her-2/neu status. Positive COX correlated with higher distant metastasis and lower local-regional control of disease. If confirmed with larger studies, these data have implications with respect to the concurrent use of COX-2 inhibitors and radiation for PMCWR.

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Oocyte maturation and ovulation are two major events that occur in fish prior to spawning. While earlier studies have shown that 17α, 20β-dihydroxy-4-pregnen-3-one (17,20β-P) and the insulin-like growth factor (IGF) system are regulators of oocyte maturation in the zebrafish (Danio rerio), it is not known whether these hormones play a role in regulating prostaglandin synthesis which is thought to mediate ovulation. I determined if 17,20β-P and human IGF-1 affect the expression of genes involved in prostaglandin biosynthesis including phospholipase A2 (cpla2) and cyclooxygenase-1/2 (ptgs1/ptgs2), or prostaglandin F2α (PGF2α) levels. 17,20β-P and IGF-1 stimulated oocyte maturation in mid-vitellogenic (MV) and full grown (FG) follicles. In FG follicles, 17,20β-P increased cpla2 expression, whereas IGF-1 increased cpla2 and ptgs2 expression. Both 17,20β-P and IGF-1 increased PGF2α production. The phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling pathways were shown to mediate IGF-1- and 17,20β-P-induced oocyte maturation and cpla2 and ptgs2 expression. Collectively, these results demonstrate that 17,20β-P and IGFs are important regulators of oocyte maturation and prostaglandin synthesis in zebrafish.